Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.

Abstract

Abstract. Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells).Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation).Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 roan. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.H ORMONE binding to the vitamin D receptor (VDR) ~ and other steroid receptor-related proteins initiates a process of receptor activation that allows the receptor to become competent to regulate transcription (7, 27). Steroid-related receptors may also mediate certain rapid (socalled nongenomic) effects of their hormonal agonists (4,17,34,68,70). Initiation of these genomic and nongenomic effects may be similar among steroid-related receptors, through biochemical and physical changes in the receptors, such as changes in phosphorylation (46, 52) and/or changes in binding to other cell components (i.e., identical proteins or other proteins [8,22,36]).Despite many studies, there is controversy concerning receptor compartmentalization and possible subcellular changes in receptor distribution patterns after binding between a steroid hormone and its receptor (44,73). Studies of steroid-related receptors in the 1960s and 1970s, prin- cipally with radioactive hormones, suggested that unstimulated receptors were mainly in the cytoplasm. Parallel studies suggested that steroid hormone binding initiated receptor translocation into the nucleus within minutes (76) or, in most studies, later than 0.5-4 h after hormone exposure (73). Subsequently, immunocytology and cell fractionation suggested that even nona...

Nucleo‐cytoplasmic cycling of the vitamin D receptor in the enterocyte‐like cell line, Caco‐2

“…In contrast, we found that the baseline GFP-VDR distribution in ROS17/2.8 (A1G) cells was predominantly cytoplasmic (>70%), a finding that confirms what others have reported for VDR distribution in ROS 17/2.8 cells [Racz and Barsony, 1999], COS-1 (Sunn et al, 2001), and COS-7 [Racz and Barsony, 1999] kidney cells, and microwave-fixed fibroblasts [Barsony et al, 1990]. Next, we observed that the signal produced by our GFP-VDR construct was diffusely distributed within the nucleus.…”

“…The deletion of VDR in the enterocyte results in a 70% reduction in intestinal calcium absorption efficiency that is coincident with a significant reduction in CaBPD9k, TRPV6, and PMCA1b gene expression [Van Cromphaut et al, 2001;Song et al, 2003a].Previous research shows that binding of 1,25(OH) 2 D 3 to VDR in the cytoplasm of cells stimulates heterodimerization of VDR with RXR and the redistribution of the VDR-RXR-hormone complex to the nucleus [Barsony et al, 1990;Michigami et al, 1999;Racz and Barsony, 1999;Sunn et al, 2001]. The nuclear import of the VDR-RXR-hormone complex is active [Racz and Barsony, 1999;Miyauchi et al, 2005], enhanced by 1,25(OH) 2 D 3 treatment [Michigami et al, 1999;Racz and Barsony, 1999;Sunn et al, 2001], dependent upon the presence of intact nuclear localization sequences (NLSs) in both VDR and RXR [Prufer et al, 2000], and requires various importins [Miyauchi et al, 2005;Yasmin et al, 2005].…”

“…For this study, cells were seeded at the density of 21,333 cell per well of an 8-chamber glass coverslip and each well was transfected with 4 µg of GFP-VDR at either 2 or 9 days post-confluence using 1:1:1 ratio of DNA/Lipofectamine/Plus Reagent. With both protocols, cells were used for experiments 2 days after transfection.Cell culture treatments-In order to avoid the effect of estrogenic agonists present in phenol red (PR) and serum on VDR distribution in living cells [Barsony et al, 1990], the cells transfected with GFP-VDR were routinely switched from their usual medium (2-day Caco-2: DMEM + 20% FBS; post-confluent Caco-2: DMEM + 10% FBS; ROS 17/2.8 (A1G) cells: Ham's F12/DMEM + 10% FBS) to PR-free medium plus 1% insulin-transferrin-selenium (ITS, Invitrogen, Carlsbad, CA) at least 24 h prior imaging session. The treatment solutions containing 100nM 1,25(OH) 2 D 3 , 100 nM Ro-26-9228 or vehicle (ethanol at final concentration of 0.01%) were prepared in this medium and kept at 37°C water bath during imaging sessions.…”

Analysis of the vitamin D system in basal cell carcinomas (BCCs)

“…9,31,[36][37][38][39][40] We have characterized the immunohistochemical staining pattern of MoAb 9A7g in BCCs. 41 All BCC samples analyzed in this study revealed strong VDR-immunoreactivity that was comparable to results that we have published previously.…”

Analysis of 1,25-Dihydroxyvitamin D3 Receptors (VDR. in Basal Cell Carcinomas

“…The monoclonal antibody 9A7␥ has been used successfully in the immunohistochemical investigation of VDR in various tissues such as chicken intestine, rat brain, disaggregated rat bone cells, rat osteosarcoma 17/2.8 cells, 23 fibroblasts, 24,25 and normal and psoriatic human skin. 9,[25][26][27] Our evaluation of 9A7␥ immunoreactivity in normal human skin is in agreement with previous observations.…”